Technical Field
The present invention relates to assays and methods of use, and more particularly, to detection of protective antigens (PA) during presymptomatic infection to eliminate the need for multiple diagnostic tests and enables early therapeutic intervention.
Related Art
Rapid presymptomatic diagnosis of Bacillus anthracis at early stages of infection plays a crucial role in prompt medical intervention to prevent rapid disease progression and accumulation of lethal levels of toxin. Bacterial infection typically alters the host's homeostasis triggering perturbations to various cellular and molecular networks. Biomarkers indicative of this altered condition can be either pathogen- or host-derived. Common targets include measurable genes, proteins, metabolites, and other indicators associated with immunological, pathological, and/or clinical outcomes [1]. Rapid detection of these biomarkers at early stages of infection increases the chances of an accurate diagnosis when the patient is presymptomatic, and successful medical intervention can be initiated. This is particularly critical for the treatment of Bacillus anthracis, the etiological bacterium of anthrax, which is often difficult to diagnose and spreads rapidly in the patient.
Clinically, anthrax presents with three different pathologies depending on the route of infection. These are cutaneous anthrax, typically identified by the appearance of a black eschar on the skin at the site of infection [2], gastrointestinal anthrax, which resembles generic food poisoning [3], and inhalational anthrax. Inhalational is the most serious form of anthrax infection and initially presents with a nonspecific prodrome resembling the flu followed by severe respiratory distress, septic shock, and death [4]. Unfortunately, other conditions present with similar symptoms, confounding an obvious diagnosis [3]. The ability of B. anthracis to form an environmentally hardened spore allows for aerosolized dispersion and has prompted its use as a bioterrorism agent [5] Inhalation of aerosolized spores results in a high percentage of morbidity and mortality due to the high exposure and infection potential. If diagnosis and proper medical intervention are not initiated early enough, the infection rapidly progresses to a point where antibiotic therapy is no longer effective due to the accumulation of lethal levels of toxin [6] and [7]. At this stage of the infection, the prognosis is poor; even with therapeutic intervention, inhalational anthrax has a mortality rate between 60 and 100% [5], [8] and [9].
Inhalational anthrax involves a complex series of host-pathogen interactions. The majority of the inhaled endospores are immediately ingested by pulmonary phagocytes and subsequently transported to the bloodstream through the lymphatic channels to the hilar and tracheobronchial lymph nodes [10], [11], [12] and [13]. During trafficking to these regional lymph nodes through the bloodstream, the endospores begin the germination process, resulting in the production of two exotoxins composed of binary combinations of protective antigen (PA)[1] and either lethal factor (LF) or edema factor (EF) [14]. These toxin proteins are analogous to other AB-type toxins and form lethal toxin (LT), from the combination of LF and PA, and/or edema toxin (ET), from the combination of EF and PA. Binding to PA occurs on the cell surface after cleavage and activation of full-length PA83 to PA63 by furin [14] and [15]. The activated PA63 conformer oligomerizes to form a heptamer that binds up to three molecules of LF, EF, or a mixture of the two [16] and [17]. The central role of PA during the intoxication process is further highlighted by its messenger RNA (mRNA) expression levels, which are reported to be 4-fold higher than LF and 14-fold higher than EF [18]. Following endocytosis of the toxin complex and subsequent acidification of the endosome, the PA63 heptamer inserts into the membrane, forming a channel through which LF and EF enter the cytosol [19]. Both toxins serve to disable the immune system. LF, a zinc-dependent endopeptidase, specifically cleaves mitogen-activated protein kinase kinases down-regulating both innate and acquired immune responses, whereas EF, an adenylate cyclase, incapacitates phagocytes and causes edema through cyclic AMP induction and accumulation of fluid [20]. The prominence of the PA fraction of the anthrax tripartite toxin and its elevated expression levels relative to the other toxin components during pathogenesis make it an ideal biomarker for diagnostic detection.
Traditional diagnosis of anthrax uses the patient's history and a battery of tests that evaluate standard morphological and phenotypic properties. These procedures involve culturing blood or cerebrospinal fluid overnight, followed by multiple hours of biochemical testing and microscopy [21]. Although definitive, the isolation of B. anthracis from biological samples is possible only late in the disease process [22]. Serological diagnosis is also an accepted technique that is sensitive and specific, but antibody responses to B. anthracis require between 8 and 12 days to develop [23]. Attempts to maintain sensitivity while reducing the time between sample collection and diagnosis have resulted in numerous genotypic identification methods using polymerase chain reaction (PCR) or reverse transcription (RT)-PCR amplification to detect anthrax-specific DNA or mRNA sequences, respectively [3]. Identification of B. anthracis infection has also been demonstrated with various immunoassays directed against key biomarkers that characterize anthrax, including spore coat antigens and toxins. These approaches are dependent on target acquisition and identification through the interaction of multiple antibodies and a chromogenic substrate [24], [25] and [26]. Unfortunately, the requirement of a chromogenic substrate limits both biomarker resolution and the detection limit (to ˜1 ng/ml). For instance, sandwich enzyme-linked immunosorbent assays (ELISAs) developed for the detection of anthrax PA exotoxin using monoclonal and polyclonal antibodies achieve the same lower PA detection limit of approximately 1 ng/ml for a detection time of more than 4 to 5 h [25] and [27]. Development of fluorescence-based assays with increased sensitivity, such as immunoassay using europium nanoparticles (NPs) [26], fluorescent covalent microspheres [28], and fluorescence resonance energy transfer (FRET) assay [29], have protein sensitivity in a range from 10 pg/ml to 60 ng/ml and an assay PA detection time of more than 4 to 5 h. Recently reported approaches for anthrax protein toxins, such as assays based on multiwall carbon nanotube sensors [30] and on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry [31], have relatively higher detection limits of 30 ng/ml and 5 pg/ml, respectively, whereas detection time, especially for the MALDI-TOF assay, is long and includes an overnight incubation stage. In essence, currently known anthrax toxin assays have a broad range of sensitivities and require multiple time-consuming incubations.
Direct detection of toxin in biological fluids following its production is an ideal approach to diagnosis because it avoids extended incubations associated with culturing and amplification. Recent advances in the field of fluorescence spectroscopy have yielded a new platform technology for fluorescence surface bioassays called microwave-accelerated metal-enhanced fluorescence (MAMEF). This technique reliably detects proteins [32], [33] and [34] and nucleic acids [35], [36] and [37] at nanogram levels and below from biological fluids within seconds. This ultra-fast technology is based on metal-enhanced fluorescence (MEF), a phenomenon that dramatically enhances chromophores' emission when in close proximity to silver NPs [38] and [39] and attached to the metal NPs. MEF is based on the specific coupling of both the ground and excited state fluorophores with the surface plasmon electrons of the proximal silver NPs, resulting in enhancement of emission and typically reducing the excited state lifetime [38] and [39]. Another component of the MAMEF technology is the use of low-power microwave irradiation of samples, which increases the rate of mass transportation and molecular diffusion, resulting in an increase in the analyte detection limit [40] and [41].
It would be advantageous to provide a detection assay system that has the ability to quickly identify non-toxic proteins in a sample before the combination of such non-toxic proteins to form a toxic protein and thereby providing early therapeutic intervention during the presymptomatic timeframe.